Transcription Protocol:

Make a second mix containing all other transcription components except protein:

4.0 ul 5X Pol III transcription buffer

0.2 ul 0.1 M DTT

0.2 ul alpha amanatin (1 mg/ml)

0.2 ul RNAse Inhibitor

H20 to a final volume of 16 microliters less the maximum volume of protein to be added.

Aliquot the second mix to all reaction tubes and incubate on ice.

Add the protein extract (typically 20-40 micrograms of a whole cell extract) and any other proteins to be added. Add extract dialysis buffer (or protein dilution buffer) to bring all reactions to the same volume.

Add 3 ul of the NTP mix to each reaction, mix by flicking the tube 5-6 times, and immediately incubate at 30 degrees (the reactions will work well between room temp and 32 degrees).

Heat 1 min. 90 degrees and put on ice. Load to 0.4 mm thick urea acrylamide gel (dont forget to clean the wells of the gel just before loading). Typically, run 300V, 60 min until the bromphenyl blue is 3/4 of the way to the bottom.